Dental caries results in 25-30 billion dollars in annual health care costs in the US and is caused by the pathogenic bacterium Streptococcus mutans. A major virulence factor is demineralization of teeth by generation of lactic acid from dietary carbohydrates and bacterial tolerance of the acid products. This is largely mediated by a multi-subunit F1F0 ATPase that spans the cytoplasmic membrane, cleaves ATP and pumps protons from the cell. In other prokaryotic and eukaryotic cells, as well as mitochondria, the same enzyme works in reverse as an ATP synthase. The signal recognition particle (SRP) pathway is a co-translational protein translocation pathway conserved in all living cells and involved in insertion and secretion of proteins into and through the cytoplasmic or endoplasmic reticulum membrane. Until it was demonstrated otherwise in S. mutans, the SRP pathway was considered essential. In S. mutans, elimination of the SRP pathway results in vulnerability to numerous stressors including acid, salt, and oxidative shock, but the bacteria generate a functional membrane and survive. Two paralogs of a family (Oxa/YidC/Alb) of highly conserved membrane-localized chaperone- insertases in mitochondria/bacteria/chloroplasts have been identified in S. mutans. Elimination of yidC2 results in a similar stress-sensitive phenotype as disruption of the SRP pathway and in both cases numerous perturbations are observed in membrane composition, including impaired ATPase activity. Elimination of yidC2 also interferes with localization and function of another key virulence factor of S. mutans, adhesin P1. Mitochondria insert key proteins into their membranes in the absence of an SRP pathway via a co-translational mechanism mediated by Oxa1 requiring a ribosome binding function of its C-terminus. S. mutans YidC2 has a similar sequence and YidC2 can replace Oxa1 in yeast implying a heretofore unrecognized co-translational translocation function of a bacterial YidC and providing an explanation for stress and acid tolerance of S. mutans lacking the SRP pathway. Conversely, the influence of YidC2 on P1 is independent of the C-terminal tail suggesting that YidC2 also serves a post-translational function and participates in the general secretion pathway that mediates translocation of surface proteins to the exterior of the cell. The goals of this proposal are 1) to use biochemical, molecular and proteomic approaches to dissect co- and post-translational pathways responsible for membrane insertion and secretion of specific substrates contributing to S. mutans virulence, 2) to establish the role of YidC2 in co-translational translocation and its interaction with streptococcal ribosomes using chemical cross-linking and immunoprecipitation, and 3) to dissect the composition and interactions among the S. mutans secretion machinery components. These objectives will advance our understanding of acid tolerance and surface biogenesis in S. mutans, suggest novel targets of therapeutic intervention, and represent a new departure in studies of membrane biogenesis and protein secretion that will further fundamental knowledge relevant to other pathogenic streptococci and Gram-positive organisms. PUBLIC HEALTH RELEVANCE: Dental caries is an infectious disease that results in 25-30 billion dollars in annual health care costs in the US and is caused by the pathogenic oral bacterium Streptococcus mutans. This organism causes tooth decay because it generates acid from dietary carbohydrates and it survives because it can tolerate its own acid end products and other harsh environmental stressors. These properties stem from the way S. mutans inserts proteins into its cytoplasmic membrane. This research will explain how proteins get into and through the membrane of this and other related pathogens.